MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Ranging from 18 to 24 nt (22 nt in general), miRNAs regulate gene expression by suppressing protein translation of target mRNAs or enhancing their degradation. miRNAs targeting mRNAs depends on the association of the miRNA/mRNA complex with Argonaute (Ago) endonuclease to form the miRNA-induced silencing complex (miRISC). We used sequencing by synthesis (SBS) deep sequencing to study the miRNAs in cultured vascular endothelial cells (ECs) exposed to hypoxia. Bioinformatics approaches were then used to analyze the hypoxia-responsive miRNAs at the genome-wide scale. Among miRNAs with greatly changed expression, Let-7s and miR-103/107 target Ago1. This result suggests that an miRNA-mediated translational de-suppression mechanism may occur in ECs under hypoxia. With these data acquired from high-throughput screening and in silico approaches, two specific aims are proposed to test the hypothesis that under hypoxia, the increased Let-7s and miR- 103/107 down-regulate Ago1. Such a suppression of Ago1 decreases miRISC-mediated miRNA targeting and hence up-regulates targeted mRNAs, which encode highly translated proteins in ECs responding to hypoxia, including vascular endothelial growth factor. Specific Aim 1 will study the Ago1-regulated miRNA/mRNA targeting in ECs responding to hypoxia. We will use Ago1 cross-linking immunoprecipitation sequencing (CLIP-seq) to profile the miRISC- associated miRNAs and their mRNA targets in ECs under normoxia and hypoxia. Specific Aim 2 will decipher the functional consequences of the miRNA-mediated translational de- suppression in cultured ECs and mouse hindlimb. Specifically, we will manipulate the expression of Let-7s and miR-103/107 in vitro and in vivo. The miRISC-mediated miRNA/mRNA targeting, mRNA-encoded proteins, and angiogenesis under normoxia and hypoxia will be examined. The elucidated mechanism will reveal mechanistic insights underlying the miRNA-regulated gene expression in ECs in response to hypoxia.